Background:The function of the AMPA receptor N-terminal domain (NTD) is unknown. Results: Deletion of the NTD leads to reduced desensitization and surface expression of GluA1-4 AMPA receptors. Conclusion:The NTD has a strong influence on the function and biosynthetic maturation of AMPA receptors. Significance: The NTD has the potential to mediate allosteric regulation of AMPA receptor signaling.

Agonist responses and channel kinetics of native α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors are modulated by transmembrane accessory proteins. Stargazin, the prototypical accessory protein, decreases desensitization and increases agonist potency at AMPA receptors. Furthermore, in the presence of stargazin, the steady-state responses of AMPA receptors show a gradual decline at higher glutamate concentrations. This “autoinactivation” has been assigned to physical dissociation of the stargazin-AMPA receptor complex and suggested to serve as a protective mechanism against overactivation. Here, we analyzed autoinactivation of GluA1–A4 AMPA receptors (all flip isoform) expressed in the presence of stargazin. Homomeric GluA1, GluA3, and GluA4 channels showed pronounced autoinactivation indicated by the bell-shaped steady-state dose response curves for glutamate. In contrast, homomeric GluA2i channels did not show significant autoinactivation. The resistance of GluA2 to autoinactivation showed striking dependence on the splice form as GluA2-flop receptors displayed clear autoinactivation. Interestingly, the resistance of GluA2-flip containing receptors to autoinactivation was transferred onto heteromeric receptors in a dominant fashion. To examine the relationship of autoinactivation to physical separation of stargazin from the AMPA receptor, we analyzed a GluA4-stargazin fusion protein. Notably, the covalently linked complex and separately expressed proteins expressed a similar level of autoinactivation. We conclude that autoinactivation is a subunit and splice form dependent property of AMPA receptor-stargazin complexes, which involves structural rearrangements within the complex rather than any physical dissociation.

AMPA receptors are glutamate-gated cation channels assembled from GluA1-4 subunits and have properties that are strongly dependent on the subunit composition. The subunits have different propensities to form homomeric or various heteromeric receptors expressed on cell surface, but the underlying mechanisms are still poorly understood. Here, we examined the biochemical basis for the poor ability of GluA3 subunits to form homomeric receptors, linked previously to two amino acid residues, Tyr-454 and Arg-461, in its ligand binding domain (LBD). Surface expression of GluA3 was improved by co-assembly with GluA2 but not with stargazin, a trafficking chaperone and modulator of AMPA receptors. The secretion efficiency of GluA2 and GluA3 LBDs paralleled the transport difference between the respective full-length receptors and was similarly dependent on Tyr-454/Arg-461 but not on LBD stability. In comparison to GluA2, GluA3 homomeric receptors showed a strong and Tyr-454/Arg-461-dependent tendency to aggregate both in the macroscopic scale measured as lower solubility in nonionic detergent and in the microscopic scale evident as the preponderance of hydrodynamically large structures in density gradient centrifugation and native gel electrophoresis. We conclude that the impaired surface expression of homomeric GluA3 receptors is caused by nonproductive assembly and aggregation to which LBD residues Tyr-454 and Arg-461 strongly contribute. This aggregation inhibits the entry of newly synthesized GluA3 receptors to the secretory pathway.␣-Amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors are tetrameric ligand-gated ion channels that mediate fast excitatory neurotransmission in vertebrate brain (1, 2). The functional properties of AMPA receptors, including channel kinetics, ion permeability, ligand pharmacology, and regulation, are determined by the subunit composition. AMPA receptors are built from four subunit types (GluA1-4), each expressed as multiple alternatively spliced and/or RNA-edited variants. Studies with native and recombinant receptors indicate that receptor assembly is not random but strongly favors certain subunit combinations (3, 4). The majority of native AMPA receptors are heteromers of edited GluA2 subunits with GluA1 or GluA3 subunits, forming channels that are impermeable to Ca 2ϩ and showing a linear current-voltage (I-V) relation (3, 5, 6). Minor native populations of homomeric AMPA receptors are formed by GluA1 or GluA4 subunits to produce inwardly rectifying and Ca 2ϩ -permeable channels (7,8).At present, the molecular logic underlying the formation and cellular processing of specific subunit assemblies in AMPA receptors is still poorly understood. The oligomerization is initiated by formation of dimers between N-terminal domains (NTD) 5 of nascent receptor polypeptides in the endoplasmic reticulum (ER) followed by interactions involving the transmembrane segments and the ligand binding domains (LBD) (9 -11). The relative strength of NTD contacts can partly explain the strong prefe...